Abstract Effective T cell co-stimulation is critical for the primary induction and subsequent specific T cell responses. In addition to increased co-inhibitory signals, insufficient co-stimulatory tumor maintenance of antigen- microenvironment accounts for a great deal of the suboptimal activation and maintenance of tumor-killing CD8 T cells. Thus, one of the major goals in the immunotherapy of cancer is to provide sustainable co-stimulatory signal to empower the generation and persistence of effective tumor-killing CD8 T cells and ultimately to achieve durable tumor control. Yet, beyond engineered CAR-T cells that contain co-stimulatory motif in the engineered TCR, means to empower sustained in situ CD8 T cell co-stimulation are still far from expectations, due to the unsustainable expression of the canonical and activation-induced family of co-stimulatory receptors on CD8 T cells in the tumor microenvironment. In this proposal, we propose to activate the constitutively expressed human CD8 T cell co-stimulatory receptor NKG2D with a novel superagonist to amplify and sustain tumor antigen-specific CD8 T cell antitumor immunity. We hypothesize that therapy with NKG2D superagonist can augment T cell-mediated immunotherapy of cancer through providing sustainable magnitude of co- stimulation to induce effective and persistent antigen-specific immune responses in tumors. We propose three Specific Aims: 1) To delineate mechanisms whereby the NKG2D superagonist provides sustainable magnitude of co-stimulation to TCR/CD3 signaling; 2) To determine the impact of the NKG2D superagonist co- stimulation on cancer therapeutic effect of adoptive T cell therapy; 3) To determine the synergistic or collaborative cancer therapeutic effect of the NKG2D superagonist co-stimulation in combination with T cell checkpoint blockades. Providing that the NKG2D superagonist is being optimized for human use, if our hypothesis is proven, the therapy can be translated into clinics in the near future to significantly improve current practice of cancer immunotherapy.